Summary

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Published: January 11, 2017
doi:

Summary

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Abstract

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

Introduction

植物产生的种子,其产生下一代。种子积累大量存储储备,如油,碳水化合物和蛋白质,对于后发芽生长。人类利用种子贮藏储备食品和动物饲料的来源,因此,植物种子是可食用的有机物全球主要供应商之一。提高种子产量是植物科学的一个重要挑战。

因为种子贮藏储量食品和工业材料的商业价值的来源,这些储备的代谢的调节的分子机制已被广泛研究1-6。进一步阐明这些机制将用于提高农作物的种子产量是有用的。发展种子植物子房受精后,他们通过一系列的发展阶段1,6,7的成熟。进一步了解的分子机制基本种子发育需要详细,要生产由一系列发育中的种子的精确基因表达谱。然而,高含量的油,蛋白质,碳水化合物,和多酚类物质在植物的种子使其难以高度纯化的RNA,这排除基因表达的精确分析隔离。

在这里,我们介绍一种从含大量油脂,蛋白质和多酚的油籽RNA提取的有效方法。使用这种方法,研究人员将能够制备高度纯化的RNA。这种RNA将成为监测的关键基因控制种子贮藏储备的开发和成熟的油籽代谢调控转录变化非常有用。

Protocol

1.从植物种子总RNA提取制备缓冲套,离心柱,1.5和2.0毫升聚丙烯管,和无核酸酶的1.5mL的聚丙烯管中。 添加1%(重量/体积)分子生物学级的聚乙烯吡咯烷酮(以下简称PVP)细胞裂解缓冲液提取RNA并涡旋大力。孵育20分钟,在25℃以完全溶解。 20分钟温育后,通过将管倒置,以防止气泡的形成轻轻混匀缓冲区。在存放使用前室温(15-25°C)。 拟南芥植物和发生在冰上1.5?…

Representative Results

我们首先调查了使用拟南芥成熟种子的PVP的最佳浓度。总RNA根据上面使用含0%,0.25%,0.5%,1.0%或2.0%的PVP细胞裂解缓冲液中描述的协议从大约1,000种子中分离。均质化和离心后,同时避免了油层和种子碎片( 图1A)收集上清液。 图1: <str…

Discussion

基因表达谱有助于我们植物生理学的理解;因此,特定的RNA分离方法已经开发了用于每个样品的条件9-12。我们调查了被RNA分离过程中从种子抑制,发现RNA结合硅胶膜是严重抑制的过程。油,蛋白质和多酚的大量抑制RNA分离。我们修改了RNA提取过程以除去这些化合物与的RNA结合到二氧化硅膜上的处理前的裂解溶液。重要的修改包括添加1%PVP(步骤1.2)和从离心裂解液上清液的收集(步骤1.7?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢功能基因组学基金和光谱法和生物成像设备,涅槃核心研究设施,以及植物型研究设施,涅槃生物资源中心的工作人员。

Materials

RNeasy Plant Mini Kit QIAGEN 74904
polyvinylpyrrolidone Sigma-Aldrich P5288-100G
HOMOGENIZER S-303 AS ONE 1-1133-02
NanoDrop Lite Thermo Scientific ND-NDL-US-CAN
PrimeScript RT reagent Kit (Perfect Real Time) TAKARA RR037A
KAPA SYBR Fast qPCR kit Kapa biosystems KK4601

References

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Cite This Article
Kanai, M., Mano, S., Nishimura, M. An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis. J. Vis. Exp. (119), e55008, doi:10.3791/55008 (2017).

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